Use of liposomes in a carrier comprising a continuous hydrophobic phase as a vehicle for cancer treatment

ABSTRACT

The invention compositions comprising a continuous hydrophobic phase and liposomes as a vehicle for delivery of an antigen capable of inducing a cytotoxic T lymphocyte (CTL) response and methods for their use in the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/674,063, filed Mar. 31, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/083,209, filed Apr. 7, 2008, now abandoned,which is a National Stage Entry of PCT/CA2006/001640, filed Oct. 5,2006, which claims the benefit and priority from U.S. ProvisionalApplication 60/806,573, filed Jul. 5, 2006, and further claims thebenefit of and priority from Canadian Patent Application Nos. 2,523,032,2,533,705; and 2,542,212, filed Oct. 7, 2005, Jan. 13, 2006, and Apr. 7,2006, respectively, the contents of each application liked herein arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present application relates to the use of a composition comprisingliposomes and a continuous hydrophobic phase as a vehicle for deliveryof an antigen capable of inducing a cytotoxic T lymphocyte (CTL)response in the treatment of cancer.

BACKGROUND OF THE INVENTION

Long lasting vaccines comprising liposomes and a variety of antigenshave been previously described in the art. These vaccine compositionshave been shown to be effective in inducing an enhanced humoral immuneresponse (determined by increased antibody production) against aspecific antigen, which is dependent on T helper 2 (Th2) function.However, for a composition to adversely affect cancer, it must be ableto induce a cell-mediated (cytotoxic T lymphocyte (CTL)) response. A CTLis a sub-group of T lymphocytes that is capable of inducing the death ofinfected somatic or tumor cells; they kill (lyse) cells that areinfected with viruses (or other pathogens), or are otherwise damaged ordysfunctional. A CTL response is mediated through T helper 1 (Th1)cytokines.

In general, CTL responses are short-lived, lasting only several weeks;(Knutson et al., Clin. Cancer. Res. 8(5) 1014-1018, 1990; Dudley et al.,J. Immunother. 24(4):363-73, 2002; and Fernando et el., Scand. J.Immunol. 47(5):459-65, 1998). Recurrence of cancer is always of concern,thus the induction of a long-lasting CTL response is necessary to ensurethat cancers do not reoccur.

Thus, there remains a need for the development of long-lastingimmune-therapeutic compositions for use in the treatment of cancer,without the need for multiple booster treatments.

SUMMARY OF THE INVENTION

In one embodiment, there is provided a composition comprising: a carriercomprising a continuous phase of a hydrophobic substance; liposomes; andat least one antigen capable of inducing a CD8⁺ cytotoxic T lymphocyte(CTL) response. The composition preferably also comprises at least one Thelper epitope.

The present application in a further aspect provides a method fortreating cancer in a subject comprising administering the compositionsas described herein.

According to another aspect, the present application provides a kituseful for treating cancer in subject comprising a composition asdescribed herein, and instructions for its use thereof

Other aspects and features of the present invention, will becomeapparent to those of ordinary skill in the art upon review of thefollowing description of specific embodiments of the invention inconjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

In the figures, which illustrate embodiments of the invention by way ofexample only:

FIG. 1 illustrates ex vivo intracellular IFN-γ staining of splenocytesfrom mice exposed to an E7 epitope of HPV 16 (R9F peptide; SEQ ID NO:1), an irrelevant peptide, or no peptide 14 days post-treatment with acomposition comprising CpG oligodeoxynucleotide (CpG ODN) (SEQ ID NO:12), and R9F peptide fused with PADRE encapsulated in liposomescontained in a PBS/FIA water-in-oil emulsion. None of the controltreatments caused expansion of CD8₊/IFN-γ T cells above backgroundlevels when exposed to R9F post-treatment (data not shown). In contrast,a 12.9 fold increase of R9F-responsive CTLs occurred in splenocytes ofmice exposed to R9F peptide as compared to mice exposed to irrelevantpeptide.

FIG. 2 illustrates lysis of R9F peptide (SEQ ID NO: 1) loaded (squares)and irrelevant peptide loaded (diamonds) EL-4 cells by splenocytes frommice treated 30 days prior with a composition comprising R9F peptidefused with PADRE (SEQ ID NO: 10), and encapsulated with CpG ODN (SEQ IDNO: 12) in liposomes suspended in a PBS/FIA water-in-oil emulsion.

FIG. 3 illustrates lysis of EL-4 cells by splenocytes from mice treatedwith one of the following compositions: (i) a fusion peptide (R9Fpeptide (SEQ ID NO: 1) fused with PADRE (SEQ ID NO: 10)) and CpG ODN(SEQ ID NO: 12) encapsulated in liposomes suspended in a PBS/FIAwater-in-oil emulsion (diamonds); (ii) unencapsulated fusion peptide andCpG ODN (SEQ ID NO: 12) in a PBS/FIA water-in-oil emulsion (trianglesand crosses), or (iii) liposome encapsulated fusion peptide in a PBS/FIAwater-in-oil emulsion (closed circles). The splenocytes were thenexposed to either R9F peptide (crosses, diamonds and closed circles) orall irrelevant peptide (SEQ ID NO: 13) (triangles and open circles) 130days post-treatment. Splenocytes from mice treated with fusion peptidealone showed base levels of lysis of EL-4 cells exposed to either R9F oran irrelevant peptide 130 days post-treatment (data not shown).

FIG. 4 illustrates the effect of a single administration of acomposition comprising CpG ODN, a fusion peptide (R9F peptide (SEQ IDNO: 1) fused with PADRE (SEQ ID NO: 10)) encapsulated in liposomessuspended in a PBS/FIA water-in-oil emulsion (open squares) on the sizeof 14-day C3 tumors in mice. Tumors were undetectable by palpation byday 30 (16 days post-treatment). In contrast, C3 tumors in mice given acontrol treatment comprising the composition as described above in theabsence of a fusion peptide (closed squares) continued to increase insize. All mice (n=10) were challenged with 0.5×10⁶ C3 tumour cells 14days prior to treatment. The difference in size of tumors in the twotreatment groups was statistically significant (p=0.002 at day 25).

FIG. 5 illustrates the effect of prophylactic treatment on thepercentage of mice that are tumor-free 61 days post-challenge with0.5×10⁶ C3 cells. Treatment groups consisted of mice treated with one ofthe following compositions comprising: (1) PBS; (2) CpG in PBS; (3)fusion peptide (R9F peptide (SEQ ID NO: 1) fused with PADRE (SEQ ID NO:10)) in PBS; (4) fusion peptide encapsulated in liposomes suspended in aPBS/FIA water-in-oil emulsion; (5) fusion peptide and CpG ODN in awater-in-oil emulsion; (6) fusion peptide and CpG ODN encapsulated inliposomes; (7) fusion peptide and the lipopeptide, Pam3Cys-SKKKK (Pam3c)encapsulated in liposomes suspended in a water-in-oil emulsion.

FIG. 6 illustrates ex vivo detection of TRP-2 specific IFN-γ producingsplenocytes (spot forming cells, SFC) in mica 8 days following a singletreatment with one of the following: (A) a composition comprising atyrosine-related protein-2 (TRP-2) peptide (S9L; SEQ ID NO: 7) withPADRE (SEQ ID NO: 10) and CpG ODN (SEQ ID NO: 12) encapsulated inliposomes suspended in a PBS/ISA51 water-in-oil emulsion; (B) acomposition comprising S9L with PADRE in liposomes suspended in awater-in-oil emulsion; or (C) a composition comprising an irrelevantpeptide (SEQ ID NO: 13) with CpG in liposomes suspended in PBS/ISA51, awater-in-oil emulsion.

FIG. 7 illustrates ex vivo detection of p53 specific IFN-γ producingsplenocytes (SFC) in mice following a single treatment with (A) amodified p53 peptide (mK9M; SEQ ID NO: 8) with PADRE (SEQ ID NO: 10) andCpG ODN (SEQ ID NO: 12) encapsulated in liposomes and suspended in aPBS/ISA51 water-in-oil emulsion, or one of the following controlcompositions: (B) mK9M with PADRE in a water-in-oil emulsion; (C) mK9Mwith PADRE encapsulated in liposomes and suspended in a water-in-oilemulsion; and (D) an irrelevant peptide (SEQ ID NO: 13) with CpG ODNencapsulated in liposomes and suspended in a water-in-oil emulsion.

FIG. 8 illustrates ex vivo detection of TRP-2- and p53-specific IFN-γproducing splenocytes (SFC) in mice following a single treatment with acomposition comprising (A) p53 (mK9M peptide; SEQ ID NO: 8) and TSP-2(V8L; SEQ ID NO: 6) peptides with PADRE (SEQ ID NO: 10) and CpG ODN (SEQID NO: 12) encapsulated in liposomes and suspended in a PBS/ISA51water-in-oil emulsion or one of the following control compositions: (B)p53 and TRP-2 peptides with PADRE encapsulated in liposomes andsuspended in a PBS/ISA51 water-in-oil emulsion; (C) p53 and TRP-2peptides with PADRE and CpG ODN; (D) p53 and TRP-2 peptides with PADRE.

FIG. 9 illustrates the effect of a single treatment with HLA-A2 E6/7 CTLepitope-containing peptides on 19-day-old TC1/A2 established tumors. Asingle treatment of a composition comprising a mixture of four HPV E6/7peptides (Y10T (SEQ ID NO: 2), L9V (SEQ ID NO: 3), T81 (SEQ ID NO: 4),and T10V (SEQ ID NO: 5)) encapsulated in liposomes with CpG ODN (SEQ IDNO: 12) and PADRE (SEQ ID NO: 10) then suspended in a PBS/ISA51water-in-oil emulsion (squares) or a long peptide containing the fourHPV E6/7 peptides described above linked together with “aay”(alanine-alanine-tyrosine) linkers (AB2 peptide; SEQ ID NO: 14)encapsulated in liposomes with CpG ODN and PADRE then suspended in awater-in-oil emulsion (diamonds) eradicated TC1/A2 tumors by 21 dayspost-treatment. Treatment with one E7 peptide of HPV (L9V; SEQ ID NO: 3)encapsulated in liposomes with CpG ODN and PADRE then suspendedwater-in-oil emulsion significantly reduced tumor size (triangles).Treatment with PBS alone (crosses) did not prevent tumor growth.

FIG. 10 illustrates tumor growth in five mice injected with PBS alone 19days post-tumor implantation.

FIG. 11 illustrates the effect of a single treatment with a compositioncomprising a mixture of four individual HLA-A2 E6/7 CTLepitope-containing peptides (Y10T, L9V, T81, and T10V; SEQ ID NOs: 2, 3,4 and 5, respectively) encapsulated in liposomes with CpG ODN (SEQ IDNO: 12) and PADRE (SEQ ID NO: 10) then suspended in a PBS/ISA51water-in-oil Emulsion on the growth of 19-day-old TC1/A2 establishedtumors in five mice.

FIG. 12 illustrates the effect of a single treatment with a compositioncomprising one long peptide (AB2; SEQ ID NO: 14) encapsulated inliposomes with CpG ODN (SEQ ID NO: 12) and PADRE (SEQ ID NO: 10) thensuspended in a PBS/ISA51 water-in-oil emulsion on the growth of19-day-old TC1/A2 established tumors in five mice.

FIG. 13 illustrates the effect of a single treatment with a compositioncomprising a HLA-A2 E7 CTL epitope (L9V; SEQ ID NO: 3) encapsulated inliposomes with CpG ODN (SEQ ID NO: 12) and PADRE (SEQ ID NO: 10) thensuspended PBS/ISA51 water-in-oil emulsion on tumor growth of 19-day-oldTC1/A2 established tumors in five mice.

FIG. 14 illustrates ex vivo detection of IFN-γ producing splenocytes inmice 9 days post-immunization by a single administration of acomposition comprising (A) four short unlinked HPV-A2 HPV E6/E7 CTLepitope-containing peptides (T8l, Y10T, L9V or T10V; SEQ ID Nos: 2, 3, 4and 5, respectively) or (B) two medium length dipeptides linked togetherwith a “kkp” linker (Y10T-kkp-L9V (SEQ ID NO: 15) and T81-kkp-T10V (SEQID NO: 16)). Both compositions were encapsulated in liposomes with CpGODN (SEQ ID NO: 12) and PADRE (SEQ ID NO: 10) then suspended in aPBS/ISA51 water-in-oil emulsion. Spleens from control mice (C) containedbackground numbers of splenocytes that could be stimulated with eachindividual short peptide or a mixture of the four short peptides toproduce IFN-γ. In contrast, spleens from mice in the group immunizedwith either the four short unlinked peptides or two medium dipeptidescontained larger numbers of splenocytes that were stimulated with thesepeptides to produce IFN-γ. Stimulation of splenocytes from miceimmunized with either formulation of the invention and stimulated withthe mixture of the four short peptides produced approximately five timesas many IFNγ-producing splenocytes compared to control splenocytesindicting the linking peptides with “kkp” had no effect on the immuneresponse.

FIG. 15 shows the growth of melanoma tumors in mice treated with one ofthe following compositions comprising at least one peptide derived froma melanoma-associated protein (TRP-2 or p53) and encapsulated with CpGODN and PADRE in liposomes suspended in a water-in-oil emulsion: (i) V8Lpeptide (SEQ ID NO: 6) (diamonds); (ii) S9L peptide (SEQ ID NO: 7)(squares); (iii) mK9M peptide (SEQ ID NO: 8) (triangles); (iv) V8L andmK9M peptides (crosses); and (v) S9L and mK9M peptides (stars). Controlmice were treated with PBS alone (circle). Mice were administered thetreatment 5 days post-tumor implantation. Each data point is the averagesize of tumors in five mice.

FIG. 16 illustrates the percentage of mice with melanoma tumors aftertreatment with one of the following compositions comprising at least onepeptides derived from a melanoma-associated protein (TRP-2 or p53) andencapsulated with CpG ODN (SEQ ID NO: 12) and PADRE (SEQ ID NO: 10) inliposomes suspended in a PBS/ISA51 water-in-oil emulsion; (i) V8Lpeptide (SEQ ID NO: 6) (diamonds); (ii) S9L peptide (SEQ ID NO: 7)(squares); (iii) mK9M peptide (SEQ ID NO: 8) (triangles); (iv) V8L andmK9M peptides (crosses); and (v) S9L and mK9M peptides (stars). Controlmice were treated with PBS alone (circles). Mice were administered thetreatment 5 days post-tumor implantation. Each data point is the averagesize of tumors in five mice.

FIG. 17 illustrates the eradication or reduction of 6-day-oldestablished B16 tumors in mice upon a single treatment with acomposition comprising CpG ODN (SEQ ID NO: 12), PADRE and a TRP-2 and/ora p53 CTL epitope, encapsulated in liposomes which were suspended in PBSthen emulsified in ISA51. A single treatment with a mixture of TRP-2 andp53 CTL epitopes rendered all mice tumor-free by 21 dayspost-administration (triangles). A single treatment with TRP-2(diamonds) or p53 (squares) alone rendered 40% of the mice tumor-free 33days post treatment. Treatment with PBS in controls mice had no effecton the progression of tumors (crosses).

FIG. 18 illustrates that C3 tumors were reduced in size in mice treatedwith a composition comprising CpG ODN (SEQ ID NO: 12) and an E7 epitopeof HPV 16 (R9F peptide; SEQ ID NO: 1) encapsulated in liposomes andsuspended in a PBS/ISA51 water-in-oil emulsion, followed by dermalapplication of Aldara™ within 15-20 hours (squares). In contrast, C3tumors were not reduced in size in control mice that received PBS(crosses) or PBS followed by dermal application of Aldara within 15-20hours to the site of PBS injection (triangles). Treatment wasadministered 5 days post tumor implantation in all treatment groups.Tumor size is the average size of tumors in ten mice.

FIG. 19 illustrates the percentage of mice having a palpable tumor aftertreatment five days post tumor implantation in all treatment groups.Only 20% of mice 20 days post-tumor implantation had a palpable tumorafter treatment with a composition comprising CpG ODN (SEQ ID NO: 12)and an E7 epitope of HPV 16 (R9F peptide; SEQ ID NO: 1) encapsulated inliposomes and suspended in a PBS/ISA51 water-in-oil emulsion, followedby dermal application of Aldara™ ointment (5% imiquimod) to the site ofadministration within 15-20 hours (diamonds). In contrast, 90% of micehad palpable tumors 20 days after treatment with PBS alone (triangles)and 100% of mice had a palpable tumor after treatment with PBS followedby a dermal application of Aldara (squares).

FIG. 20 illustrates ex vivo detection of IFN-γ producing splenocytesexposed to the melanoma-related antigen, TRP-2 (S9L peptide; SEQ ID NO:7), or B16F10 cells, 8 days post-immunization with a singleadministration of a treatment composition comprising S9L peptide and atetanus toxoid epitope (F21E; SEQ ID NO: 11) encapsulated together inliposomes with CpG ODN (SEQ ID NO: 12) in a PBS/ISA51 water-in-oilemulsion (B). Control mice were immunized with the above-describedcomposition formulated without the tetanus toxoid T helper epitope (A).Five times as many splenocytes that were stimulated with B16-F10 cellsto produce IFN-γ were present in spleens from the group immunized withthe treatment composition of the invention compared to the controlgroup.

DETAILED DESCRIPTION

The present application provides compositions comprising at least oneantigen capable of inducing a CD8⁺ cytotoxic T lymphocyte (CTL) responsetogether with at least one T helper epitope and liposomes suspended in acarrier comprising a continuous phase of a hydrophobic substance.Further, the invention teaches the use of said compositions in a methodfor treating cancer in a subject.

The compositions as described herein are useful for treating a broadrange of cancers, including, without limitation: cancers caused by humanpapilloma virus (HPV), such as, for example, cervical and/or vulvarcancer; cancers involving expression of tyrosinase, such as, forexample, melanoma; cancers involving mutations or overexpression of thep53 gene product, such as, for example, breast cancer or lymph nodemetastases; and other cancers like melanoma that express more than onetumor-associated protein simultaneously. In another embodiment, thecompositions described herein are useful for treating cancers,including, without limitation: lung, ovarian, multiple myeloma, B celllymphoma, hepatoma, sarcoma, bladder, prostate, thyroid, H/N tumors,colon, rectum, renal, pancreas, gastric, adenocarcinoma, T cellleukemia, lymphosarcoma, uterine, esophageal, non-Hodgkin's lymphomas,endometrial, and RCC tumors. Any cancer that has a cell surfacecomponent that is different in quantity or substance from the cell typefrom which the cancer is derived is a candidate for treatment by theinvention. In particular, p53 is a candidate target for broadlyapplicable cancer treatments (DeLeo, A. B., Crit. Rev. Immunol., 18:29,1998; Vierboom, M. P. M, et al., Peptide-Based Cancer Vaccines. W. M.Kast, ed. Landes Bioscience, Georgetown, 2000).

As used herein, the terms “tumor”, “tumor cells”, “cancer” and “cancercells”, (used interchangeably) refer to cells that exhibit abnormalgrowth, characterized by a significant loss of control of cellproliferation or cells that have been immortalized. The term “cancer” or“tumor” includes metastatic as well as non-metastatic cancer or tumors.A cancer may be diagnosed using criteria generally accepted in the art,including the presence of a malignant tumor.

“Treating” or “treatment of” cancer refers to an approach for obtainingbeneficial or desired results, including clinical results. Beneficial ordesired clinical results can include, but are not limited to,alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilisation of the state ofdisease, prevention of development of disease, prevention of spread ofdisease, delay or slowing of disease progression, delay or slowing ofdisease onset, amelioration or palliation of the disease state, andremission (whether partial or total). “Treating” can also meanprolonging survival of a patient beyond that expected in the absence oftreatment. “Treating” can also mean inhibiting the progression ofdisease temporarily, although more preferably, it involves halting theprogression of disease permanently in a subject.

The subject to be treated may be any vertebrate, preferably a mammal,more preferably a human.

Antigens

Suitable antigens of the composition are those that are capable ofinducing a cell-mediated (CTL) immune response in a subject.

Cell-mediated immunity is an immune response that does not involveantibodies but rather involves the activation of macrophages and naturalkiller cells, the production of antigen-specific cytotoxic T lymphocytesand the release of various cytokines in response to an antigen.Cytotoxic T lymphocytes are a sub-group of T lymphocytes (a type ofwhite blood cell) which are capable of inducing the death of infectedsomatic or tumor cells; they kill cells that are infected with viruses(or other pathogens), or are otherwise damaged or dysfunctional.

Most cytotoxic T cells express T-cell receptors that can recognise aspecific peptide antigen bound to Class I MHC molecules. These CTLs alsoexpress CDS (CD8⁺ T cells), which is attracted to portions of the ClassI MHC molecule. This affinity keeps the CTL and the target cell boundclosely together during antigen-specific activation.

Cellular immunity protects the body by, for example, activatingantigen-specific cytotoxic T-lymphocytes that are able to lyse bodycells displaying epitopes of foreign antigen on their surface, such asvirus-infected cells, cells with intracellular bacteria, and cancercells displaying tumor antigens; activating macrophages and naturalkiller cells, enabling them to destroy intracellular pathogens; andstimulating cells to secrete a variety of cytokines that influence thefunction of other cells involved in adaptive immune responses and innateimmune responses.

In one embodiment, the antigen may be, for example, a peptide, asuitable native, non-native, recombinant or denatured protein orpolypeptide, or a fragment thereof, or an epitope that is capable ofproducing a CTL immune response in a subject.

The term “polypeptide” encompasses any chain of amino acids, regardlessof length (e.g., at least 6, 8, 10, 12, 14, 16, 18 or 20 amino acids) orpost-translational modification (e.g., glycosylation orphosphorylation), and includes, for example, natural proteins, syntheticor recombinant polypeptides and peptides, epitopes, hybrid molecules,variants, homologs, analogs, peptoids, peptidomimetics, etc. A variantor derivative therefore includes deletions, including truncations andfragments; insertions and additions, for example, conservativesubstitutions, site-directed mutants and allelic variants; andmodifications, including peptoids having one or more non-amino acylgroups (for example, sugar, lipid, etc.) covalently linked to thepeptide and post-translational modifications. As used herein, the term“conserved amino acid substitutions” or “conservative substitutions”refers to the substitution of one amino acid for another at a givenlocation in the peptide, where the substitution can be made withoutsubstantial loss of the relevant function. In making such changes,substitutions of like amino acid residues can be made on the basis ofrelative similarity of side-chain substituents, for example, their size,charge, hydrophobicity, hydrophilicity, and the like, and suchsubstitutions may be assayed for their effect on the function of thepeptide by routine testing.

Polypeptides, peptides or epitopes that have substantial identity tothose disclosed herein may be used. Two sequences are considered to havesubstantial identity if, when optimally aligned (with gaps permitted),they share at least approximately 50% sequence identity, or if thesequences share defined functional motifs. In alternative embodiments,optimally aligned sequences may be considered to be substantiallyidentical (i.e., to have substantial identity) if they share at least60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity over aspecified region. The term, “identity” refers to sequence similaritybetween two polypeptides molecules. Identity can be determined bycomparing each position in the aligned sequences. A degree of identitybetween amino acid sequences is a function of the number of identical ormatching amino acids at positions shared by the sequences, for example,over a specified region. Optimal alignment of sequences for comparisonsof identity may be conducted using a variety of algorithms, as are knownin the art, including the ClustalW program, available athttp://clustalw.genome.ad.jp, the local homology algorithm of Smith andWaterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithmof Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search forsimilarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.USA 85:2444, and the computerised implementations of these algorithms(such as GAP, BESTFIT, FASTA and TFASTA is the Wisconsin GeneticsSoftware Package, Genetics Computer Group, Madison, Wis., U.S.A.).Sequence identity may also be determined using the BLAST algorithm,described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using thepublished default settings). Software for performing BLAST analysis isavailable through the National Center for Biotechnology Information(through the internet at http://www.ncbi.nlm.nib.gov/).

The amount of antigen used in a single treatment with a composition asdescribed herein may vary depending on the type of antigen and the sizeof the subject. One skilled in the art will be able to determine,without undue experimentation, the effective amount of antigen to use ina particular application. The term “effective amount” as used hereinmeans an amount effective, at dosages and for periods of time necessary,to achieve the desired result.

In one embodiment, the antigen may be at least one CTL epitope capableof inducing a CTL response. For example, the antigen may be a CTLepitope derived from a virus, such as HPV.

In another embodiment, the antigen may be a CTL epitope selected fromthe group consisting of an epitope derived from the E6 or E7 protein ofHPV.

In a further embodiment, the epitope of E6 protein of HPV comprises thepeptide sequence TIHDIILECV (T10V) (SEQ ID NO: 5). In anotherembodiment, the epitope of the E7 protein of HPV comprises a peptidesequence selected from the group consisting of RAHYNIVTF (R9F) (SEQ IDNO: 1), YMLDLQPETT (Y10T) (SEQ ID NO: 2), LLMGTLGIV (L9V) (SEQ ID NO:3), and TLGIVCPI (T81) (SEQ ID NO: 4).

In another embodiment, the CTL epitope may be an epitope of atumor-associated protein, such as for example, a melanoma-associatedprotein. In a further embodiment, the melanoma-associated protein is atyrosine related protein-2 (TRP-2) or p53, which can be obtained byvarious methods including recombinant technology or chemical synthesis.

In one embodiment an epitope of a TRP-2 derived protein comprises thepeptide sequence, for example, SVYDFFVWL (S9L; SEQ ID NO: 7). In anotherembodiment, an epitope of a TRP-2 derived protein comprises the peptidesequence VYDFFVWL (V8L; SEQ ID NO: 6). In another embodiment, an epitopeof a p53 derived protein comprises a peptide sequence selected fromKYMCNSSCM (K9M; wild type p53; SEQ ID NO: 9), KYICNSSCM (mK9M; modifiedp53; SEQ ID NO: 8), and AKXVAAWTLKAAAKYICNSSCM (mK9M (SEQ ID NO: 9)coupled to PADRE (SEQ ID NO: 10)).

In one embodiment, the composition may comprise a mixture of CTLepitopes as antigens for inducing a CTL response.

In a further embodiment, the antigen may be any peptide or polypeptidethat is capable of inducing a specific CTL response that is able toeffectively recognise a specific conformation on targeted tumor cellsand cause their destruction.

In still a further embodiment, the antigen may comprise a peptidesequence selected from the following table:

TABLE 1 Antigen Sequence HLA Patent Mart-1/ AAGIGILTV A2 U.S. Pat. No. Melan-A (SEQ ID NO: 18) 5,844,075 EAAGIGILTV A2 U.S. Pat. No. (SEQ ID NO: 19) 5,844,075 ILTVILGVL A2 U.S. Pat. No.  (SEQ ID NO: 20)5,844,057 AEEAAGIGIL B45 U.S. Pat. No.  (SEQ ID NO: 21) 7,037,509AEEAAGIGILT B45 Unknown (SEQ ID NO: 22) MCIR TILLGIFFL A2 Unknown(SEQ ID NO: 23) FLALIICNA A2 Unknown (SEQ ID NO: 24) Gp100 KTWGQYWQV A2U.S. Pat. No.  (SEQ ID NO: 25) 5,844,075 Gp100 AMLGTHTMEV A2 Unknown(SEQ ID NO: 26) MLGTHTMEV A2 Unknown (SEQ ID NO: 27) SLADTNSLAV A2U.S. Pat. No. (SEQ ID NO: 28)  5,844,075 ITDQVPFSV A2 U.S. Pat. No. (SEQ ID NO: 29) 5,844,075 LLDGTATLRL A2 U.S. Pat. No.  (SEQ ID NO: 30)5,844,075 YLEPGPVTA A2 U.S. Pat. No.  (SEQ ID NO: 31) 5,844,075VLYRYGSFSV A2 U.S. Pat. No.  (SEQ ID NO: 32) 5,844,075 RLPRIFCSC A2Unknown (SEQ ID NO: 33) LIYRRRLMK A3 Unknown (SEQ ID NO: 34) ALNFPGSQKA3 Unknown (SEQ ID NO: 35) SLIYRRRLMK A3 Unknown (SEQ ID NO: 36)ALLAVGATK A3 U.S. Pat. No.  (SEQ ID NO: 37) 6,558,671 ALLAVGATK A3U.S. Pat. No.  (SEQ ID NO: 38) 6,977,074 VYFFLPDHL A24 Unknown(SEQ ID NO: 39) SNDGPTLI Cw8 Unknown (SEQ ID NO: 40) PSA VSHSFPHPLY A1U.S. Pat. No.  (SEQ ID NO: 41) 6,037,135 FLTPKKLQCV A2 U.S. Pat. No. (SEQ ID NO: 42) 6,881,405 VISNDVCAQV A2 Unknown (SEQ ID NO: 43) PSMHSTNGVTRIY A1 Unknown (SEQ ID NO: 44) Tyrosinase KCDICTDEY A1U.S. Pat. No.  (SEQ ID NO: 45) 7,019,112 SSDYVIPIGTY A1 Unknown(SEQ ID NO: 46) Tyrosinase YMDGTMSQV A2 U.S. Pat. No.  (SEQ ID NO: 47)6,096,313 MLLAVLYCL A2 U.S. Pat. No.  (SEQ ID NO: 48) 6,291,430AFLPWHRLF A24 U.S. Pat. No.  (SEQ ID NO: 49) 6,291,430 SEIWRDIDF B44U.S. Pat. No.  (SEQ ID NO: 50) 6,291,430 MSLQRQFLR A31 U.S. Pat. No. (SEQ ID NO: 51) 5,831,016 TRP1 SVYDFFVWL A2 U.S. Pat. No. (SEQ ID NO: 52) 7,067,120 TRP2 TLDSQVMSL A2 Unknown (SEQ ID NO: 53)LLGPGRPYR A31 U.S. Pat. No.  (SEQ ID NO: 54) 5,831,016 p53 ANDPIFVVL Cw8LUnknown (SEQ ID NO: 55)

As indicated above in Table 1, proteins (polypeptides) vary in thenumber of peptide sequences that may serve as CTL-epitopes andconsequently can be used in the present invention. The following genes,without limitation, code for tumor-associated proteins that have peptidesequences that can be incorporated as antigens in the invention: p53,HPV E6 and E7, ART-4, CAMEL, CEA, Cyp-B, HER2/neu, hTERT, hTRT, iCE,MUC1, MUC2, PRAME, P15, RUI, RU2, SART-1, SART-3, WT1, PSA, tyrosinase,TRP-1, TRP-2, gp100, MART-1 /Melan A, MAGE-A1, MAGE-A2, MAGE-A3 ,MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2,GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, NASS-A, NY-ESO-1,NY-ESO-1a (CAG-3), AFP, β-catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V,G250, Ras, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m,RAGE, SART-2, TRP-2/INT2, and 707-AP.

T Helper Epitopes

T helper epitopes are a sequence of amino acids (natural or non-naturalamino acids) that have T helper activity. T helper epitopes arerecognised by T helper lymphocytes, which play an important role inestablishing and maximising the capabilities of the immune system, andare involved in activating and directing other immune cells, such ascytotoxic T lymphocytes.

A T helper epitope can consist of a continuous or discontinuous epitope.Hence not every amino acid of a T helper is necessarily part of theepitope. Accordingly, T helper epitopes, including analogs and segmentsof T helper epitopes, are capable of enhancing or stimulating an immuneresponse. Immunodominant T helper epitopes are broadly reactive inanimal and human populations with widely divergent MHC types (Celis etal. (1988) J. Immunol. 140:1808-1815; Demotz et al. (1989) J. Immunol.142:394-402; Chong et al. (1992) Infect. Immun. 60:4640-4647). The Thelper domain of the subject peptides has from about 10 to about 50amino acids and preferably from about 10 to about 30 amino acids. Whenmultiple T helper epitopes are present, then each T-helper epitope actsindependently.

In one embodiment, the composition described herein also comprises atleast one T helper epitope. In some instances, the T-helper epitope mayform part of the antigen. In particular, if the antigen is of sufficientsize, it may contain an epitope that functions as a T-helper epitope. Inother embodiments, the T-helper epitope is a separate molecule from theantigen.

In another embodiment, T helper epitope analogs may includesubstitutions, deletions and insertions of from one to about 10 aminoacid residues in the T helper epitope. T helper segments are contiguousportions of a T helper epitope that are sufficient to enhance orstimulate an immune response. An example of T-helper segments is aseries of overlapping peptides that are derived from a single longerpeptide.

Sources of T helper epitopes for use in the present invention include,for example, hepatitis B surface antigen helper T cell epitopes,pertussis toxin helper T cell epitopes, measles virus F protein helper Tcell epitope, Chlamydia trachomatis major outer membrane protein helperT cell epitope, diphtheria toxin helper T cell epitopes, Plasmodiumfalciparum circumsporozoite helper T cell epitopes, Schistosoma mansonitriose phosphate isomerase helper T cell epitopes, Escherichia coli TraThelper T cell epitopes and immune-enhancing analogs and segments of anyof these T helper epitopes.

In one embodiment, the T helper epitope is a universal T helper epitope.A universal T helper epitope as used herein refers to a peptide or otherimmunogenic molecule, or a fragment thereof, that binds to amultiplicity of MHC class II molecules in a manner that activates T-cellfunction in a class II (CD4⁺ T cells) or class I (CD8⁺ Tcells)-restricted manner.

In another embodiment, the T helper epitope may be a universal T helperepitope such as PADRE (pan-DR epitope) comprising the peptide sequenceAKXVAAWTLKAAA (SEQ ID NO: 10), wherein X may be cyclohexylalanyl. PADREspecifically has a CD4⁺ T-helper epitope, that is, it stimulatesinduction of a PADRE-specific CD4⁺ T helper response.

Tetanus toxoid has T helper epitopes that work in the similar manner asPADRE. Tetanus and diphtheria toxins have universal epitopes for humanCD4⁺ cells, (Diethelm-Okita, B. M. et al., Universal epitopes for humanCD4⁺ cells on tetanus and diphtheria toxins. J. Infect. Diseases,181:1001-1009, 2000). In another embodiment, the T helper epitope may bea tetanus toxoid peptide such as F21E comprising the peptide sequenceFNNFTVSFWLRVPKVSASHLE (amino acids 947-967; SEQ ID NO: 11).

In another embodiment, the T helper epitope is fused to at least oneantigen (i.e., a peptide), or a mixture of antigens, to make a fusionpeptide.

Carriers

The carrier of the composition comprises a continuous phase of ahydrophobic substance, preferably a liquid hydrophobic substance. Thecontinuous phase may be an essentially pure hydrophobic substance or amixture of hydrophobia substances. In addition, the carrier may be anemulsion of water in a hydrophobic substance or an emulsion of water ina mixture of hydrophobic substances, provided the hydrophobic substanceconstitutes the continuous phase. Further, in another embodiment, thecarrier may function as an adjuvant.

Hydrophobic substances that are useful in the compositions as describedherein are those that are pharmaceutically and/or immunologicallyacceptable. The carrier is preferably a liquid but certain hydrophobicsubstances that are not liquids at atmospheric temperature may beliquefied, for example by warming, and are also useful in thisinvention. In one embodiment, the hydrophobic carrier may be a PBS/FIAemulsion.

Oil or water-in-oil emulsions are particularly suitable carriers for usein the present invention. Oils should be pharmaceutically and/orimmunologically acceptable. Preferred examples of oils are mineral oil(especially light or low viscosity mineral oil), vegetable oil (e.g.,corn or canola oil), nut oil (e.g., peanut oil) and squalene. A lowviscosity mineral oil is most preferred. Animal fats and artificialhydrophobic polymeric materials, particularly those that are liquid atatmospheric temperature or that can be liquefied relatively easily, mayalso be used.

Liposomes

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single bilayer membrane) or multilamellar vesiclescharacterized by multimembrane bilayers, each bilayer may or may not beseparated from the next by an aqueous layer. A general discussion ofliposomes can be found in Gregoriadis G. Immunol. Today, 11:89-97, 1990;and Frezard, F., Braz. J. Med. Bio. Res., 32:181-189, 1999.

Although any liposomes may be used in this invention, includingliposomes made from archaebacterial lipids, particularly usefulliposomes use phospholipids and unesterified cholesterol in the liposomeformulation. The cholesterol is used to stabilize the liposomes and anyother compound that stabilizes liposomes may replace the cholesterol.Other liposome stabilizing compounds are known to those skilled in theart. For example, saturated phospholipids produce liposomes with highertransition temperatures indicating increased stability. To avoidlimiting the electrostatic association between the antigen and theliposomes, the antigen may be sequestered in the interior of theliposomes.

Phospholipids that are preferably used in the preparation of liposomesare those with at least one head group selected from the groupconsisting of phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine and phosphoinositol. More preferred are liposomes thatcomprise lipids in phospholipon 90 G. When unesterified cholesterol isalso used in liposome formulation, the cholesterol is used in an amountequivalent to about 10% of the amount of phospholipid. If a compoundother than cholesterol is used to stabilize the liposomes, one skilledin the art can readily determine the amount needed in the composition.

Liposome compositions may be obtained, for example, by using naturallipids, synthetic lipids, sphingolipids, ether lipids, sterols,cardiolipin, cationic lipids and lipids modified with poly (ethyleneglycol) and other polymers. Synthetic lipids may include the followingfatty acid constituents; lauroyl, myristoyl, palmitoyl, stearoyl,arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fattyacids.

Adjuvants

The composition may further comprise one or more pharmaceuticallyacceptable adjuvants, excipients, etc., as are known in the art. See,for example, Remington's Pharmaceutical Sciences (Remington'spharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA,1985) and The United States Pharmacopoeia: The National Formulary (USP24 NF19) published in 1999. In one embodiment, suitable adjuvantsinclude a CpG-containing oligodeoxynucleotide (CpG ODN). For example,5′-TCCATGACGTTCCTGACGTT-3′. The skilled person may select an appropriateCpG on the basis of the target species and efficacy. In place of CpG, alipopeptide, such as Pam3Cys-SKKK) (EMC Microcollections, Germany) orvariants, homologs and analogs thereof may be used. In this regard, thePam2 family of lipopeptides has been shown to be an effectivealternative to the Pam3 family of lipopeptides.

The amount of adjuvant used depends on the amount of antigen end on thetype of adjuvant. One skilled in the art can readily determine theamount of adjuvant needed in a particular application.

Compositions

In one embodiment, compositions as described herein may be formulated byencapsulating an antigen (defined as a substance that interactsspecifically with free antibody and/or with antigen-binding receptors onlymphocytes) or an antigen/adjuvant complex in liposomes to form aliposome-encapsulated antigen and mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance. If an antigen/adjuvant complex is not used in the first step,a suitable adjuvant may be added to the liposome-encapsulated antigen,to the mixture of liposome-encapsulated antigen and carrier, or to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen. The order of the process may depend on the type of adjuvantused. The resulting liposome-encapsulated antigen is then mixed with thecarrier. (It should be noted that the term “liposome-encapsulatedantigen” may refer to liposome-encapsulation of the antigen alone or tothe encapsulation of the antigen/adjuvant complex depending on thecontext.) This promotes intimate contact between the adjuvant and theantigen, and may, at least in part, account for the good immuneresponse. To facilitate use of some adjuvants, the antigen may be firstencapsulated in liposomes and the resulting liposome-encapsulatedantigen is then mixed with the adjuvant in a carrier comprising acontinuous phase of hydrophobic substance.

In formulating a composition that is substantially free of water, theantigen or antigen/adjuvant complex may be encapsulated with liposomes,which may or may not be freeze-dried, and suspended in a hydrophobicsubstance. In formulating a composition in an emulsion of water in ahydrophobic substance, the antigen or antigen/adjuvant complex may beencapsulated in liposomes, suspended in an aqueous medium followed bythe mixing of the aqueous medium with a hydrophobic substance to form anemulsion. In the case of the emulsion, to maintain the hydrophobicsubstance in the continuous phase, the aqueous medium containing theliposomes may be added in aliquots with mixing to the hydrophobicsubstance.

In one embodiment, the antigen or the liposome-encapsulated antigen maybe freeze-dried before being mixed with the hydrophobic substance orwith the aqueous medium as the case may be. In another embodiment, anantigen/adjuvant complex may be encapsulated by liposomes followed byfreeze-drying. In a further embodiment, the antigen may be encapsulatedin liposomes followed by the addition of adjuvant then freeze-drying toform a freeze-dried liposome-encapsulated antigen with externaladjuvant. In yet another instance, the antigen may be encapsulated byliposomes followed by freeze-drying before the addition of adjuvant.Freeze-drying may promote better interaction between the adjuvant andthe antigen.

In another embodiment, formulation of the liposome-encapsulated antigeninto a hydrophobic substance may also involve the use of an emulsifierto promote more even distribution of the liposomes in the hydrophobicsubstance. Typical emulsifiers are well-known in the art and includemannide oleate (Arlacel™ A), lecithin, Tween™ 80, and Spans™ 20, 80, 83and 85. The emulsifier is used in an amount effective to promote evendistribution of the liposomes. Typically, the volume ratio (v/v) ofhydrophobic substance to emulsifier is in the range of about 5:1 toabout 15:1 with a ratio of about 10:1 being preferred.

Alternatively, the antigen or antigen/adjuvant complex may be associatedwith, in contact with or separate from liposomes and not encapsulated inliposomes. The efficiency of liposome encapsulation of some hydrophilicantigens or hydrophilic antigen/adjuvant complexes may be poor so thatupon being placed in a hydrophobic environment or freeze-drying most ofthe antigen becomes associated with the external surface of theliposomes. This represents another embodiment of the invention.

In a further embodiment, an antigen (peptide or polypeptide) having aCTL epitope and PADRE (fused to the antigen or separate) may beencapsulated together in liposomes. In another embodiment, more than oneantigen may be placed together in the same liposomes. In a furtherembodiment, other substances may be in used instead of PADRE that have aT-helper epitope, for example, tetanus toxoid peptide(s). In a anotherembodiment, an adjuvant, preferably a CpG-containing ODN, may beencapsulated in the liposomes as well. The liposomes are preferablysuspended in PBS. This suspension is then emulsified in a hydrophobiccarrier, for example, ISA51 or mineral oil. The result is that liposomescontaining the antigen(s) and adjuvant(s), preferably PADRE and CpG) aresuspended in PBS which in turn is emulsified in a hydrophobic carrier,for example, ISA51 or mineral oil.

Recurrence of cancer is always of concern, thus the induction of along-lasting CTL response is important to ensure that cancers do notreoccur. In general, CTL responses are short-lived lasting only severalweeks, however, the compositions as described herein are capable ofinducing a potent CTL response that lasts for at least 30, 40, 50, 60,70, 80, 90, 100, 110, 120 or 130 days.

In one embodiment, splenocytes isolated from mice treated 130 days priorwith a composition comprising a CpG ODN and a CTL epitope of E7 proteinof HPV fused to PADRE encapsulated in liposomes suspended in awater-in-oil emulsion retained the ability to lyse mouse lymphoma EL-4cells (FIG. 3). These results indicate that compositions as describedherein are able to induce a long-lasting CTL response, which isdesirable in cancer treatment.

In one embodiment, treatment with compositions comprising CpG ODNadjuvant and TRP-2 and/or p53 peptides as antigens were able to increasethe number of antigen-specific interferon-gamma (IFN-γ) producingsplenocytes needed to combat cancer cells (FIGS. 6-8). Antigens werefused to a universal T helper epitope (PADRE) and encapsulated inliposomes suspended in a water-in-oil emulsion. Both the peptide andtumor cells expressing the protein from which the peptide was derivedwere able to induce IFN-γ production, demonstrating that use of thecomposition of invention to deliver peptide antigens resulted in animmune-response relevant to the intended target.

In another embodiment, treatment of established tumors with a singletreatment with compositions as described herein was effective insignificantly reducing tumor size and the percentage of mice with tumorspost-treatment (FIGS. 9-13 and 15).

Treatment with compositions as described herein may be followed by adermal application to the site of administration of a suitablecomposition comprising imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) or analogues thereof that are membersof a class of non-nucleoside imidazoquinolinamines (hetero-cyclic amine)that activate the immune system through localised induction ofcytokines. Imiquimod is a ligand for TLR7 and activates a Th1-likecytokine milieu that includes IFN-α, TNF-α, IL-1α, IL-6, and IL-8. In afurther embodiment, treatment with compositions as described herein maybe followed by a dermal application to the site of administration ofAldara™ ointment (imiquimod 5%) (3M, St. Paul, Minn., U.S.A.) to thesite of treatment administration.

In one embodiment, tumor size, and the percentage of tumor-bearing mice,was reduced in mice treated with a single administration of acomposition comprising CpG ODN and a fusion peptide encapsulated inliposomes with suspended in a water-in-oil emulsion, followed by adermal application of Aldara ointment at the site of treatmentadministration (FIGS. 18 and 19).

The compositions as described herein may be formulated in a form that issuitable for oral, nasal, rectal or parenteral administration.Parenteral administration includes intravenous, intraperitoneal,intradermal, subcutaneous, intramuscular, transepithelial,intrapulmonary, intrathecal, and topical modes of administration. Thepreferred routes are intramuscular, subcutaneous and intradermal toachieve a depot effect.

The compositions as described herein may be effective when administeredin a single application.

In another embodiment, the compositions as described herein may be usedin combination, before or after, other cancer therapies such asradiotherapy and chemotherapy. It has been previously shown thatmelanoma recurrence was prevented when patients diagnosed with stage IIor III melanoma were treated surgically, then given a vaccinecomposition comprising a composition to induce a CTL response tomelanoma-specific antigens. (Antonia, S. J. et al., Clin. Cancer Res.12:878-887, 2006; Allegra, C. J. and R. W. Childs, J. National CancerInst. 97:1396-1397, 2005; Cassarino, D. S. et al., J. Cutaneous Path.33:335-342, 2006; Correale, P. et al., J. National Cancer Inst.97:1437-1445, 2005; Gulley, J. L. et al., Clin. Cancer Res. 11:3353-3362, 2005; and Chakraborty, M. et al., Cancer Res. 64:4328-4337,2004).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Cellular Response

(a) Activation

To examine the specificity and rapidity of the CTL response, mice weretreated once with a composition comprising CpG ODN adjuvant, a CTLepitope of human papilloma virus (HPV) 16, namely R9F (E7 (H2-Db)peptide RAHYNIVTF, amino acids 49-57; SEQ ID NO: 1) fused to PADRE(AKXVAAWTLKAAA-OH (SEQ ID NO: 10); 50 μg/dose), which is a universal Thelper epitope, and encapsulated in liposomes (0.2 g lecithin and 0.02 gcholesterol/dose) suspended in a PBS/FIA; (phosphate bufferedsaline/Freund's incomplete adjuvant) emulsion (100 μl/dose). Fourteendays post-treatment, splenocytes (effector cells) were co-cultured for 6hours with R9F or an irrelevant peptide (KIMCNSSCM; SEQ ID NO: 13). Anex vivo intracellular IFN-65 staining of splenocytes demonstrated thatthe proportion of IFN-γ positive CD8₊ T cells (CTLs) was 13 fold higher(1.6% of splenocytes) when splenocytes were exposed to R9F peptide thanwhen splenocytes were exposed to the irrelevant peptide (0.12% ofsplenocytes or no peptide; FIG. 1). As demonstrated in FIG. 1, treatmentof mice with the above-described composition comprising R9F peptidecaused a significant expansion of CTLs that exhibit a specific responseto stimulation by HPV epitope, as compared to mice treated with acomposition comprising irrelevant peptide.

Intracellular lymphokine staining demonstrated the presence of IFN-γpositive CTLs. To demonstrate the protective function of IFN-γ producingCTLs, mice were treated with a composition comprising CpG ODN and R9Fpeptide fused with PADRE and encapsulated in liposomes suspended in awater-in-oil emulsion. Thirty days post-treatment, R9F peptide(RAHYNIVTF; SEQ ID NO: 1) loaded E4 cells (target cells; mouse lymphomacell line) and an irrelevant peptide (KIMCNSSCAM; SEQ ID NO: 13) loadedE4 cells were stimulated in vitro for 6 days with splenocytes fromtreated mice. Cytotoxicity was measured by JAM assay (FIG. 2). After the6-day in vitro stimulation, approximately 50% of R9F peptide loaded EL-4cells (Effector to Target ratio 10) were lysed by splenocytes from micetreated with the composition comprising R9F peptide (squares). Incontrast only approximately 5% of irrelevant peptide loaded EL-4 cellswere lysed by the same splenocytes (diamonds; p<0.009).

(b) Duration

The duration of the memory response induced by a single treatment with aparticular embodiment of the invention was demonstrated (FIG. 3) by thelysis of EL-4 cells by splenocytes obtained from mice 130 dayspost-treatment with the following compositions: (i) fused peptide (R9Fpeptide fused with PADRE) and CpG ODN encapsulated in liposomescontained in a PBS/FIA water-in-oil emulsion (diamonds); (ii)unencapsulated fused peptide and CpG ODN in a PBS/FIA water-in-oilemulsion (triangles and crosses); or (iii) liposome encapsulated fusedpeptide in a PBS/FIA water-in-oil emulsion without a CpG ODN adjuvant(closed circles). Splenocytes treated with liposome-encapsulated fusedpeptide, liposome-encapsulated CpG ODN, unencapsulated-fused peptide andCpG ODN, and unencapsulated-fused peptide alone served as controlsplenocytes.

The JAM assay used a six-day in vitro stimulation followed byco-culturing splenocytes (effector cells) with R9F or irrelevantpeptide-loaded EL-4 cells (target cells) that had been preloaded with³H-labelled thymidine. Splenocytes from mice immunized with fusedpeptide and CpG ODN encapsulated in liposomes suspended in awater-in-oil emulsion lysed 30% of the R9F peptide loaded target cellswhen the effector to target ratio was 25:1 and 5:1 and 15% of the targetcells when the ratio was 1:1 (FIG. 3). Splenocytes from mice given thecontrol treatment demonstrated cytotoxicity at background levels.Duration of a CTL response for >130 days following a single treatment isremarkable in relation to the duration of CTL responses reported in theliterature.

Example 2

Eradication of Cervical Cancer

Despite the development of preventative vaccines for humanpapillomavirus (HPV) induced cervical and vulvar cancer, for example,Gardasil™ and Cervarix™, a therapeutic treatment for cervical and vulvarcancer remains a high priority. In this example, a treatment compositioncomprising a CTL epitope of human papilloma virus (HPV) 16, namely R9F(E7 (H2-Db) peptide RAHYNIVTF, amino acids 49-57; SEQ ID NO: 1) was usedto induce CTLs. These CTLs need CD4⁺ T cell help for theirdifferentiation and expansion, as well as their maturation intofunctional memory CTLs. To achieve a potent CTL response through CD4⁺ Tcell help, R9F peptide was fused to the universal T helper epitope,PADRE (SEQ ID NO: 10), yielding a fusion peptide. The fused peptide wasencapsulated in liposomes together with synthetic oligodeoxynucleotidescontaining CpG ODN motifs or lipopeptide (Pam3Cys-SKKKK). Thetherapeutic composition used a PBS/FIA water-in-oil emulsion to deliverthe therapeutic formulation in a single treatment. Efficacy of thetherapeutic treatment was demonstrated using HPV 16-expressing C3 tumorcells to challenge C57BL/6 mice (10 mice/group), then treating the miceon day 14 post-challenge with the treatment composition described aboveor a control composition. By day thirty (i.e., 16 days post-treatment),complete eradication of palpable tumors was demonstrated in all 10 micein the group challenged with the C3 tumor then given treatment (FIG. 4;open squares). In contrast, tumors in all 10 control mice treated with acomposition comprising all the components of the therapeutic treatmentdescribed above except fused peptide, continued to increase in size(closed squares).

Example 3

Prophylaxis

To further demonstrate the ability of a composition of the invention toprotect against an in vivo challenge with C3 tumor cells, female C57BL/6mice were injected subcutaneously at the base of the tail with acomposition comprising R9F peptide (RAHYNIVTF; ID NO: 1) fused withPADRE (SEQ ID NO: 10) (referred to as fused peptide) and encapsulatedwith CpG ODN in liposomes suspended in a water-in-oil emulsion. Todetermine if the composition would be as protective as a compositioncomprising a replacement adjuvant to CpG ODN, mice were administered acomposite on as described above wherein CpG ODN was replaced by analternative CpG adjuvant, namely, Pam3c (Pam3Cys-sKKKK). Control groupswere injected with PBS, CpG ODN in PBS, fused peptide in PBS, fusedpeptide suspended in PBS with CpG ODN, or fused peptide encapsulated inliposomes with no adjuvant.

Fifteen days after a single treatment, 0.5×10⁶ C3 cells were implantedsubcutaneously in the left flank of treated mice as a primary challenge(FIG. 5). All mice injected with PBS or CpG ODN in PBS developed tumorswithin 2 weeks and had to be removed from the study by day 30 based ontumor size as required by animal care protocols. Treatment with fusedpeptide in PBS protected only 20% of mice (treatment group 3). Fusedpeptide encapsulated in liposomes in a PBS emulsion prevented 50% of themice from developing tumors (treatment group 4), suggesting thatliposome encapsulated fused peptide offers some protection from the C3challenge. Treatment with fused peptide and CpG ODN in a PBS emulsionprevented 60% of mice from developing tumors (treatment group 5). Incomparison, 100% of mice treated with fused peptide encapsulated inliposomes with CpG ODN remained tumor-free during the 61 daypost-challenge monitoring period (treatment group 6). To determine theduration and magnitude of the memory response directed against thetumors, mice in treatment group 6 were given a secondary challenge of6×10⁶ C3 tumor cells. All mice remained tumor-free for a further 73days, demonstrating that a single treatment of a composition of theinvention provides a robust and long-lasting cellular immune response.Similarly, replacement of CpG with Pam3c resulted in all mice remainingtumor free for 61 days (treatment group 7). These mice were notre-challenged with C3 cells.

Therapy

To evaluate treatment of established palpable C3 tumors, mice wereimplanted with 0.5×10⁶ C3 cells subcutaneously in the left flank. Oneither day 4, 5, 6 or 9 post-tumor implantation, mice (n=10) weretreated with a composition comprising CpG ODN and R9F peptide(RAHYNIVTF; SEQ ID NO: 1) fused with PADRE (SEQ ID NO: 10) (fusedpeptide) and encapsulated in liposomes and suspended in a PBS/FIAwater-in-oil emulsion a placebo (fused peptide and CpG ODN in a PBSemulsion). A single immunization eradicated tumors by day 40 in all 10mice in the treatment groups that were immunized 4, 5 or 6 dayspost-tumor implantation and all 30 mice in the group that was immunized9 days post-tumor implantation. Only one mouse maintained a tumor untilday 40 in the group treated on day 5 post-tumor implantation (Table 1).In contrast, 9/10 mice developed tumors in the groups treated with theplacebo composition on day 4 or 6 days post-tumor implantation. In thegroup of mice administered with the placebo 5 days post-challenge, 10/10mice developed tumors and 27/30 mice developed tumors in the grouptreated with the placebo vaccine on day 9 post-tumor implantation.

To evaluate whether replacement of CpG ODN with Pam3c would alter theability of a composition to eradicate C3 tumors, ten mice were treatedwith a composition comprising fused peptide encapsulated with Pam3c inliposomes and suspended in a PBS/FIA water-in-oil emulsion or a placebotreatment comprising the same composition, but without liposomes wasused to treat a second group of 10 mice. Mice in the two treatmentgroups were subsequently challenged with 1×10⁶ C3 cells in the leftflank. Therapeutic treatment of established C3 tumors using fusedpeptide encapsulated with an Pam3c in liposomes was repeated twice withsimilar results as reported in Table 1.

TABLE 2 Eradication of tumors in mice treated with a compositioncomprising R9F peptide (SEQ ID NO: 1) fused with PADRE (SEQ ID NO: 10)(fused peptide) and in liposomes and suspended in a water-in-oilemulsion containing CpG ODN or Pam3c (*) as adjuvants or a placebovaccine comprising all of the above components except liposomes. Numberof mice Treatment with tumors (day (Number of days 40 post-challengepost-tumor implantation) Placedo Treated 4  9/10 0/10 5 10/10 1/10 6 9/10 0/10 9 27/30 0/30 9* (with Pam3c)  9/10 0/10

Example 4

A Therapeutic Treatment of Melanoma

Tyrosinase is a protein overexpressed in melanoma. Peptides fromtyrosinase protein are generally poor antigens for treatment ofmelanoma. As described herein, V8L, a peptide from tyrosinase-relatedprotein (TRP-2) (amino acids 181-188; VYDFFVWL; SEQ ID NO: 6) that bindsto murine MHC, H2K² and human HLA-A2.1 was used in a therapeutictreatment to stimulate production of IFN-γ producing cells. Stimulationof the number of TRP-2 specific IFN-γ producing cells indicates that atherapeutic effect directed specifically against melanoma can beanticipated.

C57BL mice were treated once with a composition of the inventioncomprising CpG ODN and TRP-2 peptide fused to PADRE encapsulated inliposomes suspended in a water-in-oil emulsion. Control treatments werecarried out with a composition comprising liposome encapsulated TRP-2peptide with PADRE in the absence of CpG ODN, and a compositioncomprising CpG ODN and an irrelevant peptide (KIMCNSSCM; SEQ ID NO: 13)with PADRE encapsulated in liposomes. In both control treatments, theliposomes were suspended in a PBS/ISA51 water-in-oil emulsion. Ex vivodetection of IFN-γ producing splenocytes by ELISPOT indicated that thetreatment composition produced the greatest number of TRP-2 specificIFN-γ producing cells (FIG. 6, group A). The control treatment (FIG. 6,group B) produced about half as many TRP-2 specific IFN-γ producingcells and replacement of TPR-2 by an irrelevant peptide producedbackground levels of TRP-2 specific IFN-γ producing cells (FIG. 6; groupC), showing that the treatment composition of the invention produces thelargest number of TRP-2 specific IFN-γ producing cells that are requiredto combat melanoma cancer.

Example 5

A Therapeutic Treatment of Breast Cancer

The p53 gene product is an ideal and widely expressed target for therapyof malignancies, in particular, breast cancer. A large portion of humancancers exhibits p53 mutations as an early event in tumorigenesis.Overexpression of p53 is an independent predictor of more aggressivecancer, lymph node metastases, failure of standard therapeutic regimens,and ultimately of cancer-related mortality.

Mice treated with a single treatment with a composition of the inventioncomprising a modified p53 CTL epitope, mK9M, (KYICNSSCM; SEQ ID NO: 8)with CpG ODN and PADRE (SEQ ID NO: 10) encapsulated in liposomes in aPBS/ISA51 water-in-oil emulsion produced approximately 10 to 40 timesmore p53 peptide specific IFN-γ producing cells (FIG. 7; group A) thanmice treated with a composition comprising the peptide fused to PADREand CpG ODN in the absence of liposomes (FIG. 7; group B); a compositioncomprising fused peptide encapsulated in liposomes without CpG ODN; or acomposition in which the fused peptide was replaced by an irrelevantpeptide (FIG. 7; group D). Increased production of tumor specific IFN-γproducing cells is correlated with a reduction/eradication of cervicalcancer tumors (see Examples 2 and 3), therefore, one skilled in the artwould predict a similar result for p53 bearing tumors.

Example 6

Therapeutic Cancer Treatment against More than One Target

Some cancers express more than one tumor-associated protein,simultaneously. Such cancers offer more than one target for therapeutictreatment. For example, melanoma cells overexpress both p53 and TRPraising the possibility that treatments aimed at both p53 and TRPsimultaneously could be more effective and specific since cellsexpressing both p53 and TRP targets would be more vulnerable totreatment.

Mice treated with a single administration of a composition comprising amixture of p53 (mK9M; KYICNSSCM; SEQ ID NO: 8) and TRP-2 (V8L; VYDFFVWL;SEQ ID NO: 6) peptides with CpG ODN and PADRE (AKXVAAWTLKAAA, whereinX=cyclohexylalanyl); SEQ ID NO: 10) and encapsulated in liposomessuspended in a water-in-oil emulsion produced approximately equalmashers of both p53 and TRP specific IFN-γ producing cells (FIG. 8;group A). In contrast, control mice treated with a mixture of p53 fusedto PADRE and TRP-2 CTL peptides with PADRE together with CpG ODN withoutliposomes produced more TRP-2- than p53-specific IFN-γ producing cells(FIG. 8; group C), even in the absence of CpG ODN (FIG. 8; group B).Production of p53-specific IFN-γ producing cells was at levels obtainedwith the control treatments (i.e., treatment composition without CpG ODN(group B) or without CpG ODN and liposomes (group D)). These resultsindicate that mice administered the treatment composition mount atwo-pronged attack against tumors bearing TRP-2 and p53 tumor-associatedproteins. Without encapsulation of the fused peptides with CpG ODN inliposomes suspended in a water-in-oil emulsion, treated mice attack onlyone target, the TRP-2 tumor-associated protein, despite being treatedwith both TRP-2 and p53 peptides.

Example 7

HLA A2 transgenic mice were used, which have a human HLA A2 majorhistocompatibility complex (MHC) gene, and therefore express human MHC,which better mimics human cervical cancer. To be compatible with HLA A2MHC, CTL epitopes different than those used in the previous exampleswere utilized. HLA A2 Mice were treated one of the followingcompositions:

-   (1) a mixture of four E6/7 human papilloma virus (HPV) derived    peptides (MP), the sequence of each peptide being as follows:

Y10T (E7: amino acids 11-20; YMLDLQPETT; SEQ ID NO: 2); L9V(E7: amino acids 82-90; LLMGTLGIV; SEQ ID NO: 3); T81(E7: amino acids 86-93; TLGIVCPI; SEQ ID NO: 4); and T10V(E6: amino acids 29-38; TIHDILLECV; SEQ ID NO: 5);

-   (2) The above 4 peptides jollied together with “aay” linkers into a    long peptide (AB2; SEQ ID NO: 14), the sequence of which is as    follows:

TIHDIILECVaayYMLDLQPETTaayLLMGTLGIVaayTLGIVCPI;or

-   (3) A single peptide selected from the four peptides listed above,    namely L9V (E7: amino acids 82-90; LLMGTLGIV; SEQ ID NO: 3).

All treatment compositions comprised PADRE (25 μg/dose) and CpG ODN (50μg/dose) as adjuvants, and were delivered in liposomes suspended in aPBS/ISA51 water-in-oil emulsion. The mixture of 4 peptides contained 25μg of each peptide/treatment. The long peptide (AB2) was administered at100 μg/treatment. The composition comprising L9V alone contained 25μg/treatment. Control mice were injected with PBS (100 μL/treatment).

Mice were challenged with TC1/A2 tumor cells (1×10⁵ cells/mouse)implanted subcutaneously in the left flank and tumor size was measuredevery 5 days. Nineteen days post-challenge, mice (5 mice/group) weretreated with one of the above-described compositions, or injected withPBS (controls).

It is shown in FIG. 9 that a single treatment of the compositioncomprising mixture of the above-described four HPV E6/7 peptides(squares) or the AB2 long peptide (diamonds) eradicated TC1/A2 tumors 21days post-treatment. Treatment with the composition comprising the E7peptide L9V significantly reduced tumor size (triangles). Treatment withPBS alone (crosses) did not prevent tumor growth. Tumor growth wassimilar in all five mice in the control group that received PBS (FIG.10). All five mice were removed from the study on day 35 as mandated byexcessive tumor size. Tumor size is reported as the average tumor sizein five mice.

Reductions in tumor size in mice treated with composition comprising themixture of peptides was variable (FIG. 11). For example, tumor size inmouse 2 was eradicated by day 6 post-treatment. Tumors in the remainingmice were not eradicated until at least day 11 post-treatment.Reductions in tumor size in mice treated with the composition comprisingthe long peptide (AB2) were also variable (FIG. 12). However, tumorswere completely eradicated by day 21 post-treatment in all 5 mice.

Reductions in tumor size in mice treated with the composition comprisinga single HPV E7 peptide (L9V; SEQ ID NO: 3) was similar in 4/5 mice(FIG. 13). Treatment of mouse 3 did not result in tumor size reduction,suggesting that treatment with a composition comprising than one HPVpeptide either as a mixture of individual peptides or fused peptideswill protect more individuals in a population than immunization againsta single peptide.

Example 8

In example 7, four HPV 16 E6/E7 peptides were joined together to formone long peptide using the linker “aay” (-alanine-alanine-tyrosine-).This linker is hydrophobic in nature and adds to the hydrophobicity ofthe fused long peptide making peptide manufacture difficult andrequiring use of dimethyl sulphoxide to solubilize the long peptide.

In this example, a “kkp” linker (-lysine-lysine-proline-) was used inplace of the “aay” linker to form 2 dipeptides. One dipeptide wasY10T-kkp-L9V (TIHDIILECVkkpLLMGTLGIV; SEQ ID NO: 15) and the otherdipeptide was T81-kkp-T10V (TLGIVCPIkkpYMLDLQPETT; SEQ ID NO: 16). Useof the “kkp” linker resulted in hydrophilic fused peptides thatfacilitate vaccine manufacture. Using “kkp” to link peptides producedapproximately the same number of IFNγ-producing splenocytes as obtainedwhen the same four peptides were used individually (i.e., unlinked (FIG.14). These results demonstrate that use of kkp linker facilitatesmanufacture of the vaccine antigen without altering antigen processingand induction of peptide-specific IFNγ-producing splenocytes. Productionof these cells is a good indicator of effective eradication of cancercells.

Example 9

A Therapeutic Treatment against Melanoma

Examples 4, 5 and 6 demonstrate the ability of composition of theinvention to increase production of TRP-2 and p53 specific IFN-γproducing splenocytes, thereby, establishing stimulation of a cellularimmune responses against melanoma-associated proteins. B16-F10 cells(10×10³ cells/mouse) were implanted subcutaneously in the left flank ofpathogen-free C57BL/6 mice. Mice were 6-8 weeks of age at the time ofimplantation and were housed under filter top conditions with water andfood ad libitum. Five days after implantation of melanoma cells, micereceived a single treatment by subcutaneous injection of a compositioncomprising one of two peptides (V8L or S9L (SEQ ID NO: 6 or 7); 25μg/mouse) derived from TRP-2, one modified peptide (mK9M (SEQ ID NO: 8);25 μg/mouse) derived from p53 or mixtures of these peptides (25 μg ofeach peptide/mouse). All compositions also comprised both PADRE (25μg/mouse) and CpG ODN (50 μg/mouse), and delivered in liposomessuspended in a PBS/ISA51 water-in-oil emulsion Control mice received asingle administration of PBS alone. All injections ware administered atthe base of the tail. Tumor size was determined every 4-5 days using thefollowing formula: longest measurement×(shortest measurement)(Pilon-Thomas et al., J. of Immunother., 29(4), 2006).

FIG. 15 demonstrates that mice treated with a composition containingeither the V8L (SEQ ID NO: 6) (diamonds) or mK9M peptide (SEQ ID NO: 8)(triangles) inhibited growth of melanoma cells initially, but themelanoma cells overcame the initial inhibition of growth to producetumors that increased in size to 1200 mm³. Treatment with compositionscomprising S9L peptide (SEQ ID NO: 7) (squares) or mixtures of peptidesV8L, and mK9M (KYICNSSCM) (crosses), or S9L and mK9M stars) inhibitedgrowth of melanoma for the entire monitoring period. PBS alone (circles)did not have an effect on tumor growth.

Consideration of the percentage of mice that have tumors at the end ofthe study indicated that vaccine of the invention that contained eitherthe peptide mK9M (SEQ ID NO: 8) (triangles), V8L (SEQ ID NO: 6)(diamonds) or S9L (SEQ ID NO: 7) (squares) cured only 0, 40, and 40%,respectively, of the mice of their tumors (FIG. 16). In contrast,treatment With a composition comprising a mixture S9L and mK9M (stars)cured 80% of the mice of their tumors and or a mixture of V8L and mK9M(crosses) cured 100% of the mice of their tumors.

Example 10

B16 Melanoma Tumor Model

In previous examples, the efficacy of compositions of the invention wasdemonstrated in established tumors in two independent HPV-cervicalcancer models (C3 and TC1/A2). HPV-bearing tumors were eradicated bytargeting CTL epitopes of HPV presented on the surface of tumor cells.This strategy is particularly effective when treating virally inducedcancers. Tumors presenting over-expressed “self” antigens, however, aremore difficult to treat as they are invisible to the immune system. Selfantigens are tightly guarded by the tolerance mechanism. An effectivetherapeutic cancer treatment must have the ability to induce immuneresponses against over-expressed tumor-associated self antigens.Melanoma (including the B16 tumor model) is believed to down-regulateMHC class I expression and presentation of self antigens. A therapeuticcomposition for treatment of melanoma roust activate low-affinity T cellclonotypes that are capable of targeting self epitopes on the surface ofthe tumor.

A robust and specific CTL response is required for successful treatmentof melanoma by vaccination. In pre-clinical studies, it has been shownthat a B16-specific CTL activity was not sufficient to protect againstB16 tumor growth in vivo (Bellone et al., J. of Immunol.,165(5):2651-2656, 2000). Immunotherapy with CpG-matured dendritic cellspulsed with the melanoma-associated self epitope from TRP-2 failed toachieve tumor regression (Pilon-Thomas et al., J. of Immunother., 29(4),2006). In two other studies, treatment of 5-day old established B16tumors resulted in tumor eradication in less than 50% of treated miceand tumors reappeared in all treated animals (Pilon-Thomas et al., J. ofImmunother., 29(4), 2006; and Bronte et al., Cancer Res., 60:253-258,2000).

The ability of compositions of the invention to raise effective CTLresponses against multiple peptide antigens simultaneously was tested.Mice (5 mice/group) were implanted with 10⁴ B16 cells and treated once 6days post-implantation with a composition comprising (0.1 ml/dose) 25 μgof a TRP-2 CTL epitope (S9L; SVYDFFVWL SEQ ID NO: 7), 25 μg of a p53 CTLepitope (mK9M; KYICNSSCM SEQ ID NO: 8), 25 μg of PADRE and 50 μg of CpGODN per treatment. For comparison, a second group of mice (5 mice/group)were treated a composition comprising either 25 μg of the same TRP-2 CTLepitope or 25 μg of the p53 CTL epitope (K9M; KYMCNSSCM SEQ ID NO: 9),25 μg of a modified p53 CTL epitope, mK9M (SEQ ID NO: 8), 25 μg of PADRE(AKXVAAWTLKAAA, wherein X=cyclohexylalanyl; SEQ ID NO: 10) and 50 μg ofCpG ODN per treatment. All components of the composition wereincorporated in the liposomes before emulsification in the hydrophobiccarrier, ISA51. Control mice were treated with PBS alone.

A single treatment with the composition containing a mixture of theTRP-2 and p53 epitope eradicated tumors in all mice 21 dayspost-treatment (FIG. 17; triangles), whereas treatment with acomposition containing TRP-2 (diamonds) or p53 alone (squares) clearedtumors in only 40% of mice. All control mice injected with PBS developedtumors (crosses).

Example 11

Mice (C57BL/6) were challenged with C3 tumors that developed intopalpable size by 8 days post-challenge. On day 8 post-challenge, micewere divided into two control groups (10 mice/group), and a treatmentgroup that was treated with a single administration of a compositioncomprising CpG ODN and a fusion peptide (R9F peptide (SEQ ID NO: 1)fused to PADRE (SEQ ID NO: 10) encapsulated in liposomes with suspendedin a water-in-oil emulsion.

Within 15-20 hours following treatment, mice received a dermalapplication of Aldara™ ointment (25 mg (equivalent to 10-12 μl Aldara))at the site of treatment administration. The active ingredient in Aldarais imiquimod at a concentration of 5%. Imiquimod (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) is a novel synthetic compound that isa member of the imidazoquinolone family of drugs known to have theproperties of topical immune response modifiers and stimulators.Imiquimod is a ligand for TLR7 and activates a Th1-like cytokine milieuthat includes IFN-α, TNF-α, IL-1α, IL-6, and IL-8. In contrast, the twocontrol groups received either PBS alone (100 μL/mouse) or PBS followedby a dermal application of Aldara ointment (25 mg). Tumour size wasreduced in mice given the treatment composition followed by dermalapplication of Aldara (FIG. 18; crosses), but not reduced in controlsgiven PBS alone (crosses) or PBS followed by application of Aldara(triangles). FIG. 19 shows that the percentage of tumor-bearing mice wasreduced in mice given the treatment composition followed by dermalapplication of Aldara (diamonds), but not reduced in controls given PBSalone (triangles) or PBS followed by application of Aldara (squares).

Example 12

Treatment of Melanoma using Tetanus A Toxoid Peptide F21E as a T-HelperEpitope

A melanoma-related antigen, TRP-2, in combination with a T-helperepitope derived from tetanus toxoid, were encapsulated together in acomposition comprising CpG ODN in a PBS/ISA51 water-in-oil emulsion.Tetanus toxoid peptide replaced PADRE, the T-helper epitope used inprevious examples, to demonstrate that a variety of T-helper epitopescan be used. Stimulation of the number of TRP-2 peptide-specific IFN-γproducing cells indicates that a therapeutic effect directedspecifically against melanoma can be anticipated.

C57BL mice were immunized with a composition comprising a TRP-2 peptide(S9L; SEQ ID NO: 7) and the tetanus toxoid epitope F21E (amino acids947-967, FNNFTVSFWLRVPKVSASHLE; SEQ ID NO: 11) encapsulated together inliposomes with CpG ODN. Control mice were immunized with theabove-described composition formulated without the tetanus toxoid Thelper epitope. Ex-vivo detection of IFN-γ was performed by ELISPOT onsplenocytes isolated from spleens collected 8 days post-immunization.Splenocytes of control and treated mice were plated at 5×10⁵ cells perwell and were stimulated in vitro with the TRP-2 peptide (S9L), or withthe melanoma cancer cell line B16-F10 (5×10⁴ cells per well, 1:10effector to target ratio). Splenocytes of mice immunized with thetreatment composition contained the largest number of TRP-2 specificIFN-γ producing cells. The immune response was observed when splenocyteswere stimulated with the TRP-2 peptide or with the B16-F10 cells (FIG.20). Spleens from control mice demonstrated background levels of TRP-2specific IFN-γ producing cells. Thus, a strong anti-melanoma CTL immuneresponse can be detected when the splenocytes are stimulated using theTRP-2 peptide in the presence of a tetanus toxoid epitope, but alsousing the melanoma, antigens present on the surface of B16-F10 cells.

Methods

Cell Lines

The C3 cell line was maintained in Iscove Modified Dulbecco's Medium(IMDM; Sigma, St Louis, Mo.) supplemented with 10% heat-inactivatedfetal calf serum (Sigma, St Louis, Mo.), 2 mM L-glutamine (Gibco,Burlington, ON), 50 mM 2-mercaptoethanol (Gibco, Burlington, ON), 100U/ml penicillin and 100 μg/ml streptomycin (Gibco, Burlington, ON).Cells were incubated at 37° C./5% CO₂.

The EL-4 cell line is a lymphoma cell line that originated in mice. TheEL-4 cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM;Sigma, St Louis, Mo.) with high glucose content containing 2 mML-glutamine, and supplemented with 10% heat-inactivated fetal calf serum(Sigma, St Louis, Mo.), 50 mM 2-mercaptoethanol (Gibco, Burlington, ON),100 U/ml penicillin and 100 -μg/ml streptomycin (Gibco, Burlington, ON).Cells were incubated at 37° C./5% CO₂.

The B16F1 (B16) melanoma cell line was obtained from American TypeCulture Collection (ATCC), Manassas, Va.

Peptides

The HPV 16 E7 (H-2D^(b)) peptide RAHYNIVTF⁴⁹⁻⁵⁷ (R9F) containing a CTLepitope was fused to PADRE containing a CD4⁺ helper epitope by DaltonChemical Laboratories Inc. (Toronto, ON). This fusion peptide was usedat 50 μg/dose. Where indicated, R9F was used as an antigen (25 μg/dose)or in cytotoxicity assays. The peptide KYMCNSSCM (SEQ ID NO: 13)(Dalton) was used as an irrelevant control peptide.

The tyrosinase-related protein (TRP-2) peptides S9L (amino acids180-188; SVYDFFVWL; SEQ ID NO: 7) and V8L (amino acids 181-188;VYDFFVWL; SEQ ID NO: 6), as well as the p53 peptides (wild type p53(K9M), amino acids 232-240; KYMCNSSCM; SEQ ID NO: 9), modified p53peptide mK9M (amino acids 232-240; KYICNSSCM; SEQ ID NO: 8) and mK9Mcoupled to PADRE (AKXVAAWTLKAAAKYICNSSCM; SEQ ID NO: 17) were purchasedfrom Dalton Chemical Laboratories, Inc. (Toronto, ON, Canada). Thesepeptides are presented by the murine MHC-class I H-2K. S9L is alsopresented by MHC HLA A2, whereas, V8L. is not presented by MHC HLA A2.The TRP2 and p53 peptides were stored as a 1 mg/ml stock solution inDMSO. Further dilutions for vaccine manufacture were made using PBS.

All formulations of the vaccines, except those containing coupled mK9M,contained PADRE (2S μg/dose) and CpG ODN 1826 (50 μg/dose). Coupled mK9Mcontained PADRE as part of its structure, therefore, addition of freePADRE was unnecessary.

The amino acid sequence of the irrelevant peptide used in ELISPOTdeterminations was KIMCNSSCM (Dalton Chemical Laboratories Inc.).

Adjuvants

CpG ODN (Synthetic ODN 1826 with CpG motifs underlined5′-TCCATGACGTTCCTGACGTT-3′, 50 μg/dose) (SEQ ID NO: 12) was obtainedfrom Coley Pharmaceutical (Wellesley, Mass). Lipopetide (Pam3Cys-SKKKK,(100 μg/dose) was obtained from EMC Microcollections, Germany.

Treatments

Liposomes were prepared as follows; lecithin and cholesterol in a ratioof 10:1 (0.2 g lecithin end 0.02 g cholesterol/dose) were dissolved inchloroform/methanol (1:1; v/v) and the solution filter-sterilized usinga PTFE 0.2 μm filter. Chloroform and methanol were removed under reducedpressure using a rotary evaporator and traces of the solvents werefurther removed from the resulting thin lipid layer in vacuo. Forliposome encapsulation, fusion peptides with CpG were dissolved insterile PBS and the resulting solution added to the thin lipid layerwith mixing to form liposomes. The resulting suspension of liposomes wasemulsified in FIA (Sigma, St Louis, Mo.) by adding the liposome/PBSsuspension to FIA to form a water-in-oil emulsion (PBS:FIA; 1:1, v/v;100 μl/dose). In some experiments, Montanide ISA51 (Seppic, France) wasused in place of FIA as the oil carrier.

Pathogen-free C57BL/6 female mice, 6-8 weeks of age, were obtained fromCharles River Laboratories (Wilmington, Mass.) and were housed underfilter top conditions with water and food ad libitum. Institutionalanimal care and use guidelines were followed for all experiments. Micewere treated with compositions of the invention by subcutaneousinjection at the base of the tail. Unless stated otherwise, alltreatments were single administration and all treatment groups contained10 mice. Control mice were injected subcutaneously with PBS or a fusionpeptide (a selected CTL epitope fused to PADRE), R9F peptide, CpG ODNfor Pam3c), fusion peptide with CpG in PBS (100 μ1) or liposomeencapsulated fusion peptide, R9F, CpG (or Pam3c) in a water-in-oilemulsion (PBS/FIA; 1:1, v/v, 100 μl/dose).

Tumor Implantation

C3 cells used in tumor implantation were grown to 95% confluency andharvested with 0.05% trypsin. To establish tumors in mice, mice wereinjected with 0.5×10⁶ C3 cells s. c. in the left flank. Tumor sizes weredetermined every 4-5 days using the following formula: longestmeasurement×(shortest measurement)² divided by 2.

Mice (HLA A2) were challenged with TC1/A2 tumor cells (1×10⁵cells/mouse) implanted subcutaneously in the left flank. Tumor size wasmeasured every 5 days and is reported as tumor size in individual miceand as percent tumor bearing mice.

B16-F10 cells (10×10³ cells/mouse) were implanted subcutaneously in theleft flank of pathogen-free C57BL/6 mice that were 6-8 weeks of age attime of challenge. Tumor size was measured every 2-5 days and theresults reported as percent tumor bearing mice.

Cytotoxicity Assays

CTL assays, ELISPOT and intracellular staining for interferon (IFN)-γshowed the therapeutic response was specific for the selected E7 peptidesince an irrelevant peptide did not elicit CTL activity or IFN-γproduction above background. These studies indicate that increases inactivated treatment-specific cytotoxic T-cells in splenocytes from micegiven the therapeutic treatment correlate with tumor site reduction.Details of the procedures used are described below.

Lymphoblast Generation and In-Vitro Stimulation (IVS)

To examine the acute and memory CTL response, splenocytes from treatedmice were analyzed 7, 14 or 130 days post-immunization respectively,unless stated otherwise. Where stated, the cytotoxicity assay wasperformed upon one round of IVS. Briefly, three days before in vitrostimulation, naïve C57BL/6 mice were sacrificed by CO₂ asphyxiation andspleens were harvested and disassociated. Splenocytes were washed andcounted in RPMI-10 where RPMI is supplemented with 10% heat-inactivatedfetal calf serum (Sigma, St Louis, Mo.), 50 mM 2-mercaptoethanol(Gibco), 100 U/ml penicillin and 100 □g/ml streptomycin (Gibco).Splenocytes (10⁶ cells/ml) were cultured with lipopolysaccharide (25μg/ml) and dextran sulphate (7 μg/ml) treated lymphoblasts.

Syngeneic lymphoblasts were irradiated (by 4000 rad using a ¹³⁷Sc sourcefor 15 minutes) and loaded with the R9F peptide (100 μM). Peptide-loadedLPS activated lymphoblasts (3×10⁶ cells/ml) were used to stimulatesplenocytes of immunized mice in a ratio of 3:1 where effector cellswere adjusted to 3×10⁶ cells/ml, and T-stim (BD Biosciences,Mississauga, ON) was added to wells to obtain a final concentration of20%. Cells were incubated at 37° C./5% CO₂ for 6 days.

JAM Assay

EL-4 cells were labeled with 5 μCi/ml [Methyl-³H] thymidine (AmershamPharmacia, Erlangen, Germany). The cells were incubated at 37° C./5% CO₂for 24 hours then loaded with R9F or irrelevant peptides (10 μg/ml) forone hour. Suspensions of labelled target cells were then harvested,washed twice in RPMI-10, and seeded in 96-well U-bottom plates at adensity of 2×10³ cells/well. The effector cells were added by serialdilution starting at a concentration of 2×10⁵ effector cells/well. Theplates were incubated for 4 hours at 37° C./5% CO₂. The cells wereaspirated onto fiberglass filters and tritium counted using a PackardTopCount scintillation counter. The percent DNA fragmentation wascalculated using the following formula: % DNA fragmentation=(S−E)/E×100,where S is retained DNA (counts) in the absence of treatment(spontaneous) and E is retained DNA (counts) in the presence of effectorcells.

Ex Vivo Analysis of Antigen-Specific T Cells by ELISPOT

Activated antigen-specific CTLs in splenocytes harvested from treatedC57BL/6 mice were detected using a BD ELISPOT (BD Bioscience, San Diego,Calif.). Briefly, on day 7 post-treatment a 96-well nitrocellulose platewas coated with the capture antibody, a purified anti-mouse IFN-γantibody, and incubated overnight at 4° C. The antibody was discardedand the plate was blocked for 2 hours then the blocking solution wasremoved. Splenocytes were each added to their respective wells at aninitial concentration of 1 million cells/well in a final volume of 100μl followed by serial dilutions in subsequent wells of a row. Thefollowing stimulators and controls were added to 100 μl of media toobtain their desired final concentration. Either, C3 cells (5×10⁵cells/ml), the R9F peptide (10 μg/ml), the irrelevant peptide (10 μg/ml)for no peptides were added to the wells. PMA (5 ng/ml, Sigma), ionomycin(500 ng/ml, Sigma), served as positive controls and the irrelevantpeptide and media alone served as negative controls. The plate wasincubated overnight at 37° C./5% CO₂ after which the detection antibody,a biotinylated anti-mouse IFN-γ antibody, was added for 2 hours at roomtemperature. Following the intubation period, the detection antibody wasdiscarded and the enzyme conjugate (Streptavidin-HRP) was added for 1hour and lastly the plate was stained with an AEC substrate solution for20 minutes. The plate was washed and left to air dry overnight forvisualization of spots using a magnifying lens.

Intracellular Cytokine Staining (ICS)

Splenocytes retrieved from spleens of tumor-free mice as previouslydescribed, washed twice with RPMI-10 (500×g, 5 minutes) and resuspendedin RMPI-10 (10×10⁶ cells/ml). Splenocytes (1×10⁶ cells/well) were addedto wells of a 96-well flat bottom plate and incubated with R9F or anirrelevant peptide at a final concentration of 3 μg/ml in duplicatecolumns for each peptide. In experiments that used EL-4 cells todemonstrate the protective function of IFNγ-producing CD8⁺ CTLs, EL-4cells (1×10⁵ cells/well) loaded with either R9F or the irrelevantpeptide were incubated for 6 hours at 37° C./5% CO₂ before cytotoxicitymeasurements.

Intracellular cytokine staining was performed as described in theCytofix™/Cytoperm™ kit instruction manual (BD Biosciences, Mississauga,ON). In brief, after addition of stimulants, GolgiStop was added to eachwell and the plates were incubated (37° C./5% CO₂) for 4 hours. Cellswere washed with staining buffer then incubated (20 minutes at 4° C., inthe dark) with anti-CD8 serum, washed again with staining bufferfollowed by incubation with anti-IFN-γ (30 minutes at 4° C. in thedark). This was followed by washes with perm/wash buffer after whichcells were resuspended with perm/wash buffer and transferred to FACStubes (BD Falcon). Staining was assessed by FACSCalibur (BD Biosciences,San Jose, Calif.), and data were analysed using CellQuest software.

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All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

The invention claimed is:
 1. A method for inducing an enhanced cytotoxicT lymphocyte (CTL) response thereby treating cancer or inhibiting orpreventing the growth or proliferation of cancer cells in a subject inneed of cancer treatment comprising: encapsulating at least one antigencomprising a CTL epitope and at least one T helper epitopes inliposomes; combining said liposomes with a carrier consistingessentially of oil or a water-in-oil emulsion to form a composition; andadministering said composition to said subject.
 2. The method of claim1, wherein the cancer is selected from the group consisting of:cervical, vulvar, melanoma, breast, lung, ovarian, multiple myeloma, Bcell lymphoma, hepatoma, sarcoma, bladder, prostate cancer, thyroid, H/Ntumors, colon, rectal, renal, pancreatic, gastric, adenocarcinoma, Tcell leukemia, lymphosarcoma, uterine, esophageal, non-Hodgkin'slymphomas, endometrial, and RCC tumors.
 3. The method of claim 1,wherein said T helper epitope is a separate molecule from said antigen.4. The method of claim 1, wherein said antigen comprises said T helperepitope or wherein said antigen is fused to said T helper epitope. 5.The method of claim 1, wherein said antigen comprises a combination ofCTL epitopes.
 6. The method of claim 1, wherein said CTL epitope isderived from a tumor-associated protein.
 7. The method of claim 1,wherein said CTL epitope is derived from a virus.
 8. The method of claim1, wherein said liposomes are freeze-dried before they are combined withsaid carrier.
 9. The method of claim 1, further comprising encapsulatingan adjuvant in said liposomes or combining an adjuvant with saidcarrier.
 10. The method of claim 1 wherein the oil is mineral oil, nutoil, or squalene.
 11. The method of claim 1, wherein the subject is amammal.
 12. The method of claim 1, wherein the subject is a human. 13.The method of claim 1, wherein the liposome comprises a phospholipid.14. The method of claim 13, wherein the phospholipid has at least onehead group selected from phosphoglycerol, phosphoethanolamine,phosphoserine, phosphocholine and phosphoinositol.
 15. The method ofclaim 1, wherein the liposome comprises unesterified cholesterol. 16.The method of claim 1, wherein the liposome comprises lipids inphospholipon 90 G.
 17. The method of claim 1, wherein the liposomecomprises lipids obtained from archaebacterial bacteria.
 18. The methodof claim 1, wherein the liposome comprises natural lipids, syntheticlipids, sphingolipids, ether lipids, sterols, cardiolipin, cationiclipids, or lipids modified with poly (ethylene glycol) or otherpolymers.
 19. The method of claim 18, wherein the synthetic lipidsinclude fatty acid constituents selected from lauroyl, myristoyl,palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, and erucoyl, orcombinations thereof.
 20. The method of claim 1, wherein CTL epitope isderived from human papilloma virus.
 21. The method of claim 1, whereinthe CTL epitope is an epitope of an E6 or E7 protein of human papillomavirus (HPV).
 22. The method of claim 21, wherein the E6 epitopecomprises the peptide sequence TIHDIILECV (T10V; SEQ ID NO: 5).
 23. Themethod of claim 1, wherein the antigen comprises a plurality of CTLepitopes linked to form a single polypeptide.
 24. The method of claim 6,wherein the tumor associated protein is p53.
 25. The method of claim 24,wherein the tumor-associated protein is tyrosinase-relatedprotein-2(TRP-2).
 26. The method of claim 1, wherein the antigencomprises a p53 epitope and a TRP-2 epitope.
 27. The method of claim 26,wherein the p53 epitope comprises the peptide sequence KYICNSSCM (mK9M)(SEQ ID NO: 8).
 28. The method of claim 26, wherein the TRP-2 epitopecomprises the peptide sequence SVYDFFVWL (SEQ ID NO: 7) or the peptidesequence VYDFFVWL (SEQ ID NO: 6).
 29. The method of claim 1, wherein theT helper epitope is a universal T helper epitope, PADRE (pan-DR epitope)(SEQ ID NO: 10), or F21E (SEQ ID NO: 11).
 30. The method of claim 1,wherein the T helper epitope is fused to the at least one antigen. 31.The method of claim 9, wherein the adjuvant is a CpGoligodeoxynucleotide (CpG ODN) a lipopeptide, or Pam3Cys-SKKKK.
 32. Amethod for inducing an enhanced cytotoxic T lymphocyte (CTL) responsethereby treating cancer or inhibiting or preventing the growth orproliferation of cancer cells in a subject in need of cancer treatmentcomprising administering a composition comprising: a carrier consistingessentially of oil or a water-in-oil emulsion; liposomes; at least oneantigen comprising a CTL epitope; and at least one T helper epitope;wherein the antigen and the T helper epitope are encapsulated withinsaid liposomes.
 33. The method of claim 32, wherein the cancer isselected from the group consisting of: cervical, vulvar, melanoma,breast, lung, ovarian, multiple myeloma, B cell lymphoma, hepatoma,sarcoma, bladder, prostate cancer, thyroid, H/N tumors, colon, rectal,renal, pancreatic, gastric, adenocarcinoma, T cell leukemia,lymphosarcoma, uterine, esophageal, non-Hodgkin's lymphomas,endometrial, and RCC tumors.
 34. The method of claim 32, wherein said Thelper epitope is a separate molecule from said antigen.
 35. The methodof claim 32, wherein said antigen comprises said T helper epitope orwherein said antigen is fused to said T helper epitope.
 36. The methodof claim 32, wherein said antigen comprises a combination of CTLepitopes.
 37. The method of claim 32, wherein said CTL epitope isderived from a tumor-associated protein.
 38. The method of claim 32,wherein said CTL epitope is derived from a virus.
 39. The method ofclaim 32, wherein said liposomes are freeze-dried before they arecombined with said carrier.
 40. The method of claim 32, furthercomprising encapsulating an adjuvant in said liposomes or combining anadjuvant with said carrier.
 41. The method of claim 32, wherein thesubject is a mammal.
 42. The method of claim 32, wherein the subject isa human.
 43. The method of claim 32, wherein the liposome comprises aphospholipid.
 44. The method of claim 43, wherein the phospholipid hasat least one head group selected from phosphoglycerol,phosphoethanolamine, phosphoserine, phosphocholine and phosphoinositol.45. The method of claim 32, wherein the liposome comprises unesterifiedcholesterol.
 46. The method of claim 32, wherein the liposome compriseslipids in phospholipon 90 G.
 47. The method of claim 32, wherein theliposome comprises lipids obtained from archaebacterial bacteria. 48.The method of claim 32, wherein the liposome comprises natural lipids,synthetic lipids, sphingolipids, ether lipids, sterols, cardiolipin,cationic lipids, or lipids modified with poly (ethylene glycol) or otherpolymers.
 49. The method of claim 48, wherein the synthetic lipidsinclude fatty acid constituents selected from lauroyl, myristoyl,palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, and erucoyl, orcombinations thereof.
 50. The method of claim 32, wherein CTL epitope isderived from human papilloma virus.
 51. The method of claim 32, whereinthe CTL epitope is an epitope of an E6 or E7 protein of human papillomavirus (HPV).
 52. The method of claim 51, wherein the E6 epitopecomprises the peptide sequence TIHDIILECV (T10V; SEQ ID NO: 5).
 53. Themethod of claim 32, wherein the antigen comprises a plurality of CTLepitopes linked to form a single polypeptide.
 54. The method of claim37, wherein the tumor associated protein is p53.
 55. The method of claim54, wherein the tumor-associated protein is tyrosinase-related protein-2(TRP-2).
 56. The method of claim 32, wherein the antigen comprises a p53epitope and a TRP-2 epitope.
 57. The method of claim 56, wherein the p53epitope comprises the peptide sequence KYICNSSCM (mK9M) (SEQ ID NO: 8).58. The method of claim 56, wherein the TRP-2 epitope comprises thepeptide sequence SVYDFFVWL (SEQ ID NO: 7) or the peptide sequenceVYDFFVWL (SEQ ID NO: 6).
 59. The method of claim 32, wherein the Thelper epitope is a universal T helper epitope, PADRE (pan-DR epitope)(SEQ ID NO: 10), or F21E (SEQ ID NO: 11).
 60. The method of claim 32,wherein the T helper epitope is fused to the at least one antigen. 61.The method of claim 40, wherein the adjuvant is a CpGoligodeoxynucleotide (CpG ODN) a lipopeptide, or Pam3Cys-SKKKK.